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Temperature-mediated FOXO1 transport dependent on RANBP2, XPO1 and IPO7 SUMOylation. a Left: Confocal images of H1 ESCs with annotated treatments; FOXO1, DAPI were co-stained with RANBP2, Importin-7 (IPO7) or Exportin-1 (XPO1); right: Nuclear FFOXO1 versus cytosolic or whole-cell FFOXO1 (n = 10, 10 and 12 images from 5 experiments for RANBP2, IPO7 and XPO1 RNA interference, respectively). b Confocal images of proximity ligation assay (PLA) on proteins of interest in H1 ESCs at indicated conditions. c Average counts of PLA puncta per cell from (b) (from left to right, n = 4, 4, 5, 7, 5 and 5 images from 3 experiments). d Up: immunoblots of XPO1, IPO7 and FOXO1 in total protein extracts (Input) and FOXO1 immunoprecipitated (IP-ed) fractions from H1 cells at annotated conditions; down: signal intensities normalized (n = 4 experiments). e Left: confocal images of PLA on proteins of interest in H1 ESCs at indicated conditions; right: average counts of PLA puncta per cell (from left to right, n = 4, 6, 6 and 6 experiments). f Up: immunoblots of XPO1 and IPO7 in Input and SUMO1 IP-ed fractions from H1 cells at annotated conditions; down: signal intensities normalized (n = 5 experiments). g Left: confocal images of PLA on FOXO1 interaction with endogenous IPO7 (n = 13 images from 3 experiments), or with overexpressed, HA-tagged IPO7 with a K-R mutation on its SUMOylation site in ARPE-19(WT) cells (n = 12 images from 3 experiments); WT, wild-type; right: average counts of PLA puncta per cell. Data are shown as mean and SEM. Statistics: one-way ANOVA followed by Tukey’s test (a, g) and two-tailed Student’s t test (c−f). Scale bars: 20 μm (a, b, e) and 2.5 μm (g). Source data are provided as a Source Data file.
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